Stabilization of thrombocytes at ambient temperatures

ABSTRACT

Provided herein are formulations and methods for the stabilization of one or more thrombocytes at ambient temperatures. Also provided are formulations and methods for the stabilization of one or more thrombocytes in an inactivated state in a blood sample at ambient temperatures. Further provided are articles of manufacture and kits and methods for substantially stable storage of one or more thrombocyte at ambient temperatures.

CROSS-REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 15/316,677, filed Dec. 6, 2016, which is a U.S.National Phase Application of International Patent Application No.PCT/US2015/034967, filed Jun. 9, 2015, which claims the benefit of U.S.Provisional Application No. 62/010,151, filed Jun. 10, 2014, all ofwhich are incorporated by reference herein in their entirety.

BACKGROUND OF THE INVENTION 1. Technical Field

The present invention relates generally to stabilization of one or morethrombocytes at ambient temperatures. In particular, the inventionrelates to formulations, compositions, articles of manufacture, kits andmethods for substantially stable storage of one or moremetabolically-active thrombocytes at ambient temperatures.

BACKGROUND

Whole blood is a complex mixture of cells, nucleic acids, proteins andvarious other analytes. In particular, blood components include, but arenot limited to: cells, such as leukocytes (monocytes, lymphocytes andgranulocytes), erythrocytes, thrombocytes and circulating tumor cells;nucleic acid molecules, such a circulating-free DNA (cfDNA);polypeptides, such as lipoproteins, albumin and serum proteins, andother various analytes.

Thrombocytes or platelets are anucleated cells that play a key role inthe clotting of blood. Thrombocytes are small, disc-like cells thatcirculate in mammalian blood and are involved in hemostasis.Thrombocytes secrete a wide variety of growth factors that assist inpromoting blood clotting and tissue regeneration.

The level of circulating thrombocytes in a healthy individual iscontrolled within a physiological range of about (150-400)×103 per mm³.Suboptimal levels of thrombocytes (thrombocytopenia) can lead toexcessive bleeding, whereas levels exceeding optimal concentrations canlead to the formation of thromboli (blood clots) that can obstruct bloodvessels and can lead to higher risk of stroke, pulmonary embolus ormyocardial infarction.

Circulating thrombocytes are typically present in an inactivated state,and are maintained in the inactivated state by factors produced byendothelial cells lining the blood vessel lumen. Upon disruption orinjury to this endothelial layer, thrombocytes come in contact withcollagen or von Wildebrand's factor, which activates the thrombocytescausing the thrombocytes to aggregate (i.e., clot). This activation andaggregation also may occur by the enzymatic activity of thrombin or inthe presence of ADP. Upon activation, thrombocytes release the contentsof alpha and dense granules that include growth factors and fibrinogenthat assist in clot formation and help promote recruitment offibroblasts to promote wound healing. Activated thrombocytes can bedistinguished from inactivated thrombocytes by their morespherical/stellate shape.

The activation, aggregation and/or release of numerous growth factorsand other intracellular components of thrombocytes during the collectionof whole blood can greatly hinder the quantitation and analysis of thesecells. The addition of various anti-coagulants to maintain aninactivated thrombocytes at ambient temperatures results in only about13-52% inactivated thrombocytes at 24 hours making accurate quantitativeanalysis of total thrombocytes essentially impossible at this timepoint. Thus, there exists a need for improved formulations for andmethods of stabilizing thrombocytes at ambient temperatures for a timesufficient for storage and shipping thrombocytes for research,diagnostic and therapeutic purposes.

SUMMARY OF THE INVENTION

The formulations, compositions and methods of the present inventionadvantageously provide for the stabilization of thrombocytes at ambienttemperatures and these cells remain functional and retain the ability tobe activated post-blood collection for a period of at least 24 hours,significantly increasing the time for storage and shipping ofsubstantially stable thrombocytes for research, diagnostic and potentialtherapeutic applications. Disclosed herein in some embodiments, areformulations for substantially stable storage of one or morethrombocytes at ambient temperatures, wherein the one or morethrombocytes are stabilized for a period of at least six hours. In someembodiments, the one or more thrombocytes are stabilized in aninactivated state. In some embodiments, the one or more thrombocytes arein a blood sample. In some embodiments, the one or more thrombocytes arein an inactivated state in a blood sample. In some embodiments, the oneor more thrombocytes are isolated from a blood sample. In someembodiments, at least 90% of the thrombocytes are stabilized in aninactivated state for a period of at least six hours. In someembodiments, at least 90% of the thrombocytes are stabilized in aninactivated state for a period of at least nine hours. In someembodiments, the thrombocytes are stabilized in an inactivated state fora period of at least 7 hours, at least 8 hours, at least 9 hours, atleast 10 hours, at least 11 hours, at least 12 hours, at least 13 hours,at least 14 hours, at least 15 hours, at least 16 hours, at least 17hours, at least 18 hours, at least 19 hours, at least 20 hours, at least21 hours, at least 22 hours, at least 23 hours or at least 24 hours. Insome embodiments, the formulation comprises: (i) a pH buffer; (ii) ananti-coagulant; (iii) at least one non-reducing sugar or polyol; and(iv) a functionalized carbohydrate. In some embodiments, the formulationcomprises a polyol selected from the group consisting of glycol,glycerol, erythritol, threitol, arabitol, xylitol, ribitol, adonitol,mannitol, sorbitol, galactitol, fucitol, iditol and inositol, andcombinations thereof. In some embodiments, the polyol is a pentosepolyol or a hexose polyol. In some embodiments, the pentose polyol isadonitol. In some embodiments, the functionalized carbohydrate issucralfate or sucrose octasulfate. In some embodiments, thefunctionalized carbohydrate is sucrose octasulfate. In some embodiments,the non-reducing sugar is sucrose or trehalose. In some embodiments, thenon-reducing sugar is trehalose. In some embodiments, the anticoagulantis EDTA or hirudin. In some embodiments, the pH buffer is 2×phosphatebuffered saline or Tris-HCl.

In one aspect of the invention, formulations are provided forsubstantially stable storage of one or more thrombocytes in aninactivated state in a blood sample at ambient temperatures, wherein theone or more thrombocytes are stabilized in an inactivated state for aperiod of at least six hours. In some embodiments, at least 90% of thethrombocytes are stabilized in an inactivated state for a period of atleast six hours. In some embodiments, the thrombocytes are stabilized inan inactivated state for a period of at least 7 hours, at least 8 hours,at least 9 hours, at least 10 hours, at least 11 hours, at least 12hours, at least 13 hours, at least 14 hours, at least 15 hours, at least16 hours, at least 17 hours, at least 18 hours, at least 19 hours, atleast 20 hours, at least 21 hours, at least 22 hours, at least 23 hoursor at least 24 hours. In certain embodiments, the formulation comprises(i) a pH buffer, (ii) an anticoagulant, (iii) at least one non-reducingsugar or polyol, and (iv) a functionalized carbohydrate. In someembodiments, the polyol is selected from the group consisting of glycol,glycerol, erythritol, threitol, arabitol, xylitol, ribitol, adonitol,mannitol, sorbitol, galactitol, fucitol, iditol inositol, andcombinations thereof. In some embodiments the polyol is a pentose polyolor a hexose polyol. In some embodiments, the polyol is adonitol. In someembodiments, the functionalized carbohydrate is sucralfate or sucroseoctasulfate. In some embodiments, the functionalized carbohydrate issucrose octasulfate. In some embodiments, the non-reducing sugar issucrose or trehalose. In some embodiments, the non-reducing sugar istrehalose. In some embodiments, the anticoagulant is EDTA or hirudin. Insome embodiments, the anticoagulant is EDTA, the functionalizedcarbohydrate is sucralfate or sucrose octasulfate, and the non-reducingsugar is sucrose or trehalose. In yet other embodiments, theanticoagulant is EDTA, the functionalized carbohydrate is sucroseoctasulfate and the non-reducing sugar is trehalose. In someembodiments, the pH buffer is 2×phosphate buffered saline or Tris-HCl.Disclosed herein, in some embodiments are formulations for substantiallystable storage of one or more thrombocytes at ambient temperatures,comprising a halogenated disaccharide derivative and an anticoagulant,wherein the one or more thrombocytes are stabilized for a period of atleast six hours. In some embodiments, the one or more thrombocytes arestabilized in an inactivated state. In some embodiments, the one or morethrombocytes are in a blood sample. In some embodiments, the one or morethrombocytes are stabilized in an inactivated state in a blood sample.In some embodiments, the one or more thrombocytes are isolated from ablood sample. In some embodiments, the anticoagulant is hirudin. In someembodiments, the halogenated disaccharide derivative is selected fromthe group consisting of sucralose(1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside),trichloronated maltose,1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-6-O-monododecanoate-α-D-galactopyranoside,1,6-sichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-6-O-monotetradecanoate-α-D-galactopyranoside,and combinations thereof. In some embodiments, the formulation consistsessentially of hirudin and sucralose(1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside).

In some embodiments, there are provided formulations for substantiallystable storage of one or more thrombocytes in an inactivated state in ablood sample at ambient temperatures, comprising a halogenateddisaccharide derivative, wherein the one or more thrombocytes arestabilized in an inactivated state for a period of at least six hours.In some embodiments, the halogenated disaccharide derivative preferablyis selected from the group consisting of sucralose(1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside),trichloronated maltose,1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-6-O-monododecanoate-α-D-galactopyranosideand1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-6-O-monotetradecanoate-α-D-galactopyranoside,and more preferably the halogenated disaccharide derivative is sucralose(1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside).In some embodiments, the formulations further comprise an anticoagulant,preferably hirudin. In some embodiments, the anticoagulant is hirudin.In some embodiments, the formulation consists essentially of hirudin andsucralose(1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside).

Disclosed herein in some embodiments, are compositions of substantially,stably stored one or more thrombocytes comprising one or morethrombocytes admixed with a disclosed formulation. In some embodiments,the one or more thrombocytes are in a blood sample. In some embodiments,the one or more thrombocytes are isolated thrombocytes. In someembodiments, the one or thrombocytes are in an inactivated state.

Disclosed herein in some embodiments, are articles of manufacture,comprising a formulation described herein contained within a bloodcollection tube. In some embodiments, the blood collection tube is anevacuated blood collection tube.

Disclosed herein, in some embodiments, are kits comprising an article ofmanufacture described herein and a package insert.

Disclosed herein in some embodiments, are methods for substantiallystable storage of one or more thrombocytes at ambient temperatures,comprising: admixing the one or more thrombocytes from a subject with aformulation provided herein, wherein the one or more thrombocyte isstabilized for a period of at least six hours. In some embodiments, theone or more thrombocytes are stabilized in an inactivated state. In someembodiments, the one or more thrombocytes are in a blood sample from thesubject. In some embodiments, the one or more thrombocytes arestabilized in an inactivated state in a blood sample from the subject.In some embodiments, the one or more thrombocytes are isolated from ablood sample from the subject. In some embodiments, at least 90% of thethrombocytes are stabilized in an inactivated state for a period of atleast six hours. In some embodiments, at least 90% of the thrombocytesare stabilized in an inactivated state for a period of at least ninehours. In some embodiments, the method further comprises activating theone or more thrombocyte in an inactivated state, by the addition of anactivating agent to promote thrombocyte aggregation. In someembodiments, the activating agent is ADP. In some embodiments, themethod further comprises activating the one or more thrombocyte by theaddition of an activating agent to promote thrombocyte aggregation. Insome embodiments, the activating agent is ADP. In some embodiments, thesubject is an animal. In some embodiments, the subject is a mammal. Insome embodiments, the mammal is a human.

Disclosed herein in some embodiments, are methods for substantiallystable storage of one or more thrombocyte in an inactivated state in ablood sample at ambient temperatures, comprising, admixing a bloodsample from a subject with a formulation provided herein, wherein theone or more thrombocytes are stabilized in an inactivated state for aperiod of at least six hours. In some embodiments, at least 90% of thethrombocytes are stabilized in an inactivated state for a period of atleast six hours. In some embodiments, the thrombocytes are stabilized inan inactivated state for a period of at least 7 hours, at least 8 hours,at least 9 hours, at least 10 hours, at least 11 hours, at least 12hours, at least 13 hours, at least 14 hours, at least 15 hours, at least16 hours, at least 17 hours, at least 18 hours, at least 19 hours, atleast 20 hours, at least 21 hours, at least 22 hours, at least 23 hoursor at least 24 hours. In some embodiments, the blood sample is admixedwith the stabilization formulation at the time the blood sample iscollected from the subject to substantially stabilize the one or morethrombocytes in the inactived state post collection from the subject. Insome embodiments, the method further comprises activating the one ormore thrombocytes by the additional of an activating agent. In someembodiments, the activating agent is ADP. In further embodiments of themethods, the subject is an animal, more preferably a mammal, and evenmore preferably a human.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to formulations, compositions, articles ofmanufacture, kits, and methods for substantially stable storage of oneor more thrombocyte at ambient temperatures. In some embodiments, theone or more thrombocytes are stored in an inactivated, but activatable,state in a blood sample. In one aspect, the formulations describedherein beneficially maintain the integrity of inactivated, metabolicallyactive, thrombocytes that may be subsequently analyzed for activation orthat may be used in therapeutic applications for promoting bloodclotting in a patient.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” includes one or more methods, and/or steps of the type describedherein which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, or ±5%, or even ±1% from the specifiedvalue, as such variations are appropriate for the disclosed compositionsor to perform the disclosed methods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents, patent applicationsand publications referred to herein are incorporated by reference intheir entirety.

Formulations are provided, in some embodiments, for substantially stablestorage of metabolically-active thrombocytes at ambient temperatures. Insome embodiments, the thrombocytes are isolated from a blood sample. Insome embodiments, the thrombocytes are in a blood sample. In someembodiments, the thrombocytes are inactivated. In certain embodiments,the thrombocyte stabilization formulations comprise a pH buffer, ananticoagulant, a non-reducing sugar, a polyol, and a functionalizedcarbohydrate. In certain other embodiments, the thrombocytestabilization formulations comprise a pH buffer, an anticoagulant, apolyol and a functionalized carbohydrate. In certain other embodiments,the stabilization formulations comprise a pH buffer, an anticoagulant, anon-reducing sugar, and a functionalized carbohydrate. In still yetanother embodiment, the stabilization formulations comprise ahalogenated disaccharide derivative and an anticoagulant. In still yetanother embodiment, the stabilization formulations comprise ahalogenated disaccharide derivative, an anticoagulant, and a pH buffer.The formulations are capable of stabilizing at least 60%, 70%, 80% oreven 90% inactivated, metabolically-active thrombocytes in a bloodsample at ambient temperatures for a period of at least 6 hours, or atleast 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, atleast 11 hours, at least 12 hours, at least 13 hours, at least 14 hours,at least 15 hours, at least 16 hours, at least 17 hours, at least 18hours, at least 19 hours, at least 20 hours, at least 21 hours, at least22 hours, at least 23 hours or at least 24 hours.

The term “ambient temperature” as used herein refers to common indoorroom temperatures. In some embodiments, ambient temperature is 15 to 32°C. In some embodiments, ambient temperature is 20 to 27° C.

In another aspect of the present invention, formulations are providedfor substantially stable storage of inactivated, metabolically-activethrombocytes in a blood sample at ambient temperatures. In certainembodiments, the thrombocyte stabilization formulations comprise a pHbuffer, an anticoagulant, a non-reducing sugar, a polyol, and afunctionalized carbohydrate. In certain other embodiments, thethrombocyte stabilization formulations comprise a pH buffer, ananticoagulant, a polyol, and a functionalized carbohydrate. In certainother embodiments, the stabilization formulations comprise a pH buffer,an anticoagulant, a non-reducing sugar, and a functionalizedcarbohydrate. In still yet another embodiment, the stabilizationformulations comprise a halogenated disaccharide derivative and ananticoagulant. In still yet another embodiment, the stabilizationformulations comprise a halogenated disaccharide derivative, ananticoagulant, and a pH buffer. The formulations are capable ofstabilizing at least 60%, 70%, 80% or even 90% inactivated,metabolically-active thrombocytes in a blood sample at ambienttemperatures for a period of at least 6 hours, or at least 7 hours, atleast 8 hours, at least 9 hours, at least 10 hours, at least 11 hours,at least 12 hours, at least 13 hours, at least 14 hours, at least 15hours, at least 16 hours, at least 17 hours, at least 18 hours, at least19 hours, at least 20 hours, at least 21 hours, at least 22 hours, atleast 23 hours or at least 24 hours.

In another aspect, compositions are provided herein in which a bloodsample is admixed with a thrombocyte stabilization formulation toproduce substantially stable one or more inactivated thrombocytes in awhole blood preparation. In still other embodiments, a compositioncomprising purified or substantially purified one or more thrombocyteadmixed with a stabilization formulation of the present invention areprovided.

Formulation Reagents

A. pH Buffers

According to certain embodiments, the herein described formulations andcompositions for substantially stable storage of one or morethrombocytes include one or more pH buffers. In some embodiments, the pHbuffer is any of a large number of compounds known in the art for theirability to resist changes in the pH of a solution, such as in an aqueoussolution in which the pH buffer is present. Selection of one or moreparticular pH buffers for inclusion in a stable storage composition maybe done based on the present disclosure and according to routinepractices in the art, and may be influenced by a variety of factorsincluding the pH that is desired to be maintained, the nature of thebiological sample, the solvent conditions to be employed, the othercomponents of the formulation to be used, and other criteria. Forexample, typically a pH buffer is employed at a pH that is within about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 pH unit of a protondissociation constant (pKa) that is a characteristic of the buffer.

Non-limiting examples of pH buffers include citric acid, tartaric acid,malic acid, sulfosalicylic acid, sulfoisophthalic acid, oxalic acid,borate, CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), CAPSO(3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid), EPPS(4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid), HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid), MOPS(3-(N-morpholino)propanesulfonic acid), MOPSO(3-morpholino-2-hydroxypropanesulfonic acid), PIPES(1,4-piperazinediethanesulfonic acid), TAPS(N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid), TAPSO(2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid),TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), bicine(N,N-bis(2-hydroxyethyl)glycine), tricine(N-[tris(hydroxymethyl)methyl]glycine), tris(tris(hydroxymethyl)aminomethane) and bis-tris(2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol). Insome embodiments, including any of those set forth in Table 1, have a pHof about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0.

B. Polyols

Also as described herein, certain embodiments include at least onepolyol in the composition for substantially stable storage of viable,inactivated thrombocytes in a whole blood sample at ambienttemperatures. Polyols are polyhydric alcohols containing two or morehydroxyl groups and have the general formula H(CHOH)_(n)H, wherein n isan integer selected from 2 to 7 inclusive. Polyols differ in chainlength with most polyols having five- or six carbon chains being derivedfrom pentoses (five-carbon sugars) and hexoses (six-carbon sugars);however shorter and longer carbon chain polyols also exist. Exemplarypolyols include, but are not limited to, glycol, glycerol, erythritol,threitol, arabitol, xylitol, ribitol, adonitol, mannitol, sorbitol,galactitol, fucitol, iditol and inositol. Selection of one or moreparticular polyols for inclusion in a substantially stable storagecomposition may be done based on the present disclosure and according toroutine practices in the art, and may be influenced by a variety offactors including other formulation components. In certain embodiments,the polyol present in the formulation is a pentose polyol. In someembodiments, the polyol is adonitol. In some embodiments, the polyol ispresent at a concentration between 20-100 mM, or between about 25-75 mM.In some embodiments, the polyol is a pentose polyol and is present at aconcentration between 20-100 mM, or between about 25-75 mM. In someembodiments, the polyol is adonitol and is present at a concentrationbetween 20-100 mM, or between about 25-75 mM.

C. Disaccharide Derivatives

In certain embodiments, the formulations or compositions forsubstantially stable storage of one or more inactivated thrombocyte in awhole blood sample at ambient temperatures, including those in Table 1,include at least one halogenated disaccharide derivative. In someembodiments, the halogenated disaccharide derivative is a di- ortri-chlorinated disaccharide. In some embodiments, such di- ortri-chlorinated disaccharides unexpectedly are capable of substantiallystable storage of inactivated thrombocytes either alone or in thepresence of only a buffer. Halogenated disaccharide derivatives areknown, e.g., see US Patent Publication No. 2014/0065062, and includesucralose(1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside),trichloronated maltose,1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-6-O-monododecanoate-α-D-galactopyranoside,and1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-6-O-monotetradecanoate-α-D-galactopyranoside.Selection of one or more particular halogenated disaccharide derivativefor inclusion in a substantially stable storage composition may be donebased on the present disclosure and according to routine practices inthe art, and may be influenced by a variety of factors including otherformulation components. In some embodiments, the functionalizedcarbohydrate is sucralose and is present at about 1.0-50.0 mM. In someembodiments, the functionalized carbohydrate is sucralose and is presentat about 10.0-30.0 mM. In some embodiments, the functionalizedcarbohydrate is sucralose and is present at about 25.0 mM.

D. Functionalized Carbohydrates

In some embodiments described herein, the formulations, including thosein Table 1, include a functionalized carbohydrate. Exemplaryfunctionalized carbohydrates include sucralfate or sucrose octasulfateand it will be appreciated that from the present disclosure the skilledperson may select other functionalized carbohydrates for use in a stablestorage formulations and compositions for viable, activatable,thrombocytes, as may vary based on the other components of thecomposition that are employed. In some embodiments, the concentration offunctionalized carbohydrates in the present formulations andcompositions, including those set forth in Table 1, is about 0.005-1.0mM. In some embodiments, the concentration of functionalizedcarbohydrates in the present formulations and compositions, includingthose set forth in Table 1, is about 0.25-0.5 mM.

E. Non-Reducing Sugars

In some embodiments, the formulations and compositions for substantiallystable storage of thrombocytes at ambient temperatures include at leastone non-reducing sugar. In some embodiments, the formulations andcompositions for substantially stable storage of viable, inactivatedthrombocytes in a whole blood sample at ambient temperatures include atleast one non-reducing sugar. As used herein, “non-reducing sugars”refers to carbohydrate molecules that lack a functional aldehyde group.Exemplary non-reducing sugars include sucrose and trehalose. In someembodiments, the non-reducing sugar is sucrose. In some embodiments, thenon-reducing sugar is trehalose. In some embodiments, the trehalose ispresent at a concentration of about 1.0-50 mM. In some embodiments, thetrehalose is present at a concentration of about 10.0-30 mM. In someembodiments, the trehalose is present at a concentration of about 25 mM.

F. Anticoagulants

In some embodiments, an anticoagulant is included in the presentlydescribed formulations or compositions. Such anticoagulants are known inthe art. Exemplary anticoagulants include ethylenediaminetetraaceticacid (EDTA), hirudin, heparin, and sodium citrate. In some embodiments,the anticoagulant is hirudin. In some embodiments, the hirudin ispresent at a concentration of about 1.0-50 μg/mL. In some embodiments,the hirudin is present at a concentration of about 1.0-25 μg/mL. In someembodiments, the hirudin is present at a concentration of about 10-20μg/mL.

Exemplary Formulations for Stabilization of Thrombocytes at AmbientTemperatures

In some embodiments, the formulations, compositions and methods of thepresent invention advantageously provide for the substantially stablestorage of thrombocytes at ambient temperatures for a period of at leastsix hours. In some embodiments, the formulations, compositions andmethods of the present invention advantageously provide for thesubstantially stable storage of thrombocytes in their naturalcirculating, inactivated state in a blood sample at ambienttemperatures, wherein the cells retain the ability to be activated postcollection for a period of at least six hours. In other embodiments, atleast 90% of the thrombocytes are stabilized in an inactivated state fora period of at least six hours. In other embodiments, the thrombocytesare stabilized in an inactivated state for a period of at least 7 hours,at least 8 hours, at least 9 hours, at least 10 hours, at least 11hours, at least 12 hours, at least 13 hours, at least 14 hours, at least15 hours, at least 16 hours, at least 17 hours, at least 18 hours, atleast 19 hours, at least 20 hours, at least 21 hours, at least 22 hours,at least 23 hours or at least 24 hours.

In certain embodiments, the formulations for the substantially stablestorage of thrombocytes at ambient temperatures comprise a pH buffer, ananticoagulant, a non-reducing sugar, a polyol, and a functionalizedcarbohydrate. In certain embodiments, the stabilization formulationscomprise a pH buffer, an anticoagulant, a polyol, and a functionalizedcarbohydrate. In certain embodiments, the formulations for thesubstantially stable storage of thrombocytes comprise a pH buffer, ananticoagulant, a non-reducing sugar, and a functionalized carbohydrate.In some embodiments, the formulations for the substantially stablestorage of thrombocytes comprise a halogenated disaccharide derivative,and an anticoagulant, and may further comprise a pH buffer. In someembodiments, the anti-coagulant is sprayed and dried on the bloodcollection tube, container, or vessel prior to collection of the bloodsample from the subject. In some embodiments, the anti-coagulant isadded directly to the formulations described herein.

In certain embodiments, the pH buffer is Tris-HCl, the polyol is apentose alcohol, the non-reducing sugar is trehalose, the anticoagulantis EDTA or hirudin, and the functionalized carbohydrate is sucroseoctasulfate. In certain embodiments, the pH buffer is Tris-HCl, thepolyol is adonitol, the non-reducing sugar is the D+ isomer oftrehalose, the anticoagulant is EDTA or hirudin, and the functionalizedcarbohydrate is sucrose octasulfate. In certain embodiments, the pHbuffer is Tris-HCl, the polyol is adonitol, the non-reducing sugar isthe D+ isomer of trehalose, the anticoagulant is hirudin, and thefunctionalized carbohydrate is sucrose octasulfate. In some embodiments,the disaccharide derivative is a halogenated disaccharide and theanticoagulant is hirudin. In some embodiments, the halogenateddisaccharide is sucralose and the anticoagulant is hirudin.

In some embodiments, the formulations for the substantially stablestorage of inactivated thrombocytes at ambient temperatures include theexemplary formulations provided in Table 1.

TABLE 1 Exemplary Formulations for Stabilizing Inactivated,Metabolically-active Thrombocytes in a Human Blood Sample at AmbientTemperatures Tris- Sucrose HCl Adonitol Trehalose Octasulfate SucraloseFormulation (mM) (mM) (mM) (mM) (mM) A 2.5 100 1.0 B 2.5 50 25.0 1.0 C2.5 25.0 1.0 D 2.5 50 25.0 0.5 E 2.5 25 F 25

Methods for Preparing Exemplary Formulations

In some embodiments, the exemplary Formulations A-F of Table 1 areprepared using materials commercially available from suppliers andpreparing such formulations is accomplished using the methods disclosedherein as well as other methods known to those skilled in the art.

In some embodiments, pre-weighed solid components are added to asuitable vessel, such as a square bottle, to which the aqueouscomponents are added. The reaction mixture is agitated, e.g., byshaking, until the solid components have completely dissolved and thenthe pH of the mixture is adjusted to the desired pH using a suitableacid, e.g., hydrochloric acid. The resulting formulations are thensterilized, e.g., using a 0.22 micron filter, and stored at roomtemperature.

In one example, a 50 mL preparation of a 20× Formula A is prepared asfollows: 15.2034 gr of adonitol (Calbiochem, catalogue #121739) and1.251 g sucrose octasulfate potassium salt (Toronto Research Chemicals,catal,ogue #S69900) are added to a square bottle. A 30 mL volume ofwater is added, followed by the addition of 2.5 mL of Tris-HCl(Invitrogen, catalogue #15567-027). Additional water is added to qc theformulation to a final total volume of 50 mL. The mixture is shakenuntil fully dissolved, and hydrochloric acid is added to adjust the pHto 7.51. The solution is sterile filtered (0.22 μm pore size) undervacuum to yield the resulting formulation.

In another example, a 50 mL preparation of a 20× Formula B is preparedas follows: 7.5994 g of adonitol, 9.4997 g D-(+)-trehalose dihydrate(Fluka, catalogue #90210), and 1.25 g sucrose octasulfate potassium saltare added to a square bottle. A 30 mL volume of water is added, followedby the addition of 2.5 mL of Tris-HCl (Invitrogen, catalogue#15567-027). Additional water is added to qc the formulation to a finaltotal volume of 50 mL. The mixture is shaken until fully dissolved, andhydrochloric acid is added to adjust the pH to 7.51. The solution issterile filtered (0.22 μm pore size) under vacuum to yield the resultingformulation.

In another example, a 50 mL preparation of a 20× Formula C is preparedas follows: 9.5003 g D-(+)-trehalose dehydrate and 1.2502 g sucroseoctasulfate potassium salt are added to a square bottle. A 30 mL volumeof water is added, followed by the addition of 2.5 mL of Tris-HCl(Invitrogen, catalogue #15567-027). Additional water is added to qc theformulation to a final total volume of 50 mL. The mixture is shakenuntil fully dissolved, and hydrochloric acid is added to adjust the pHto 7.52. The solution is sterile filtered (0.22 μm pore size) undervacuum to yield the resulting formulation.

In another example, a 50 mL preparation of a 20× Formula D is preparedas follows: 7.5994 g of adonitol, 9.5003 g D-(+)-trehalose dihydrate and0.625 g sucrose octasulfate potassium salt. A 30 mL volume of water isadded, followed by the addition of 2.5 mL of Tris-HCl (Invitrogen,catalogue #15567-027). Additional water is added to qc the formulationto a final total volume of 50 mL. The mixture is shaken until fullydissolved, and hydrochloric acid is added to adjust the pH to 7.51. Thesolution is sterile filtered (0.22 μm pore size) under vacuum to yieldthe resulting formulation.

In another example, a 50 mL preparation of a 20× Formula E is preparedas follows: 9.9997 g of sucralose (Sigma, catalogue #69293) is added toa square bottle. A 30 mL volume of water is added, followed by theaddition of 2.5 mL of Tris-HCl (Invitrogen, catalogue #15567-027).Additional water is added to qc the formulation to a final total volumeof 50 mL. The mixture is shaken until fully dissolved and hydrochloricacid is added to adjust the pH to 7.54. The solution is sterile filtered(0.22 μm pore size) under vacuum to yield the resulting formulation.

In another example, a 50 mL preparation of a 20× Formula F is preparedas follows: 9.9993 g of sucralose (Sigma, catalogue #69293) is added toa square bottle. Water is added to qc the formulation to a final totalvolume of 50 mL. The mixture is shaken until fully dissolved, providinga solution having a pH of 7.54. The solution is sterile filtered (0.22μm pore size) under vacuum to yield the resulting formulation.

Purified Stabilized Thrombocytes

In some embodiments, the substantially stabilized one or morethrombocytes in a blood sample at ambient temperatures are purifiedusing well known methods employed by those skilled in the art. Apparatusand kits for purifying thrombocytes from blood are well known (e.g., seeU.S. Pat. Nos. 5,234,593; 6,315,706 and 7,708,152). In certainembodiments, the thrombocytes are purified using a bag PC (plateletconcentrate) prepared after collection of blood in a bag and theapheresis PC obtained by the use of a component blood collecting device.These methods separate thrombocytes from blood using centrifugalseparation. In some embodiments, substantially stabilized, intact,metabolically active viable cells are advantageously purified byaffinity chromatography or by fluorescence activated cell sorting (FACS)analysis using antibodies generated against a native wild type membraneproteins and receptors, and which method is not possible using otherstorage formulations that denature these cellular proteins.

In some embodiments, the purified one or more thrombocyte aresubsequently stored in the formulations described herein for extendedperiods before analysis or use.

Articles of Manufacture

In certain embodiments, articles of manufacture are provided, whichcomprise a formulation provided herein, contained within a suitableblood collection tube, container or vessel. In some embodiments, theformulation is selected from those set forth in Table 1. In someembodiments, these articles of manufacture are used for substantiallystable storage of one or more blood component by stabilizing one or moreblood component at the time of blood collection. In certain embodiments,the blood collection tube is an evacuated blood tube having less thanatmospheric pressure to withdraw a predetermined volume of whole blood.In some embodiments, these articles of manufacture are used in the kitsand methods described herein.

Kits

In certain embodiments, there are provided kits comprising any one ofthe articles of manufacture described herein and a package insert. Insome embodiments, the components of the kit are supplied in a packagingmeans, such as a compartmentalized plastic enclosure, preferably with ahermetically sealable cover so that the contents of the kit can besterilized and sealed for storage.

Methods for Substantially Stable Storage of One or More Thrombocyte in aBlood Sample at Ambient Temperatures

Described herein, in some embodiments, are methods for substantiallystable storage of one or more thrombocyte at ambient temperatures. Insome embodiments, the methods are for substantially stable storage ofone or more thrombocytes in an inactivated state in a blood sample atambient temperatures.

In certain embodiments, the methods comprise admixing a blood samplewith a formulation for substantially stable storage of one or morethrombocyte at ambient temperatures for a period of at least six hours.In some embodiments, the one or more thrombocytes are isolated from ablood sample. In some embodiments, the one or more thrombocytes arestabilized in an inactivated state. In some embodiments, at least 90% ofthe thrombocytes remain in an inactivated state for a period of at leastsix hours. In other embodiments, the thrombocytes are stabilized in aninactivated state for a period of at least 7 hours, at least 8 hours, atleast 9 hours, at least 10 hours, at least 11 hours, at least 12 hours,at least 13 hours, at least 14 hours, at least 15 hours, at least 16hours, at least 17 hours, at least 18 hours, at least 19 hours, at least20 hours, at least 21 hours, at least 22 hours, at least 23 hours or atleast 24 hours. In certain embodiments, the formulation is one of theformulations set forth in Table 1.

In certain embodiments, the methods comprise admixing a blood samplewith a formulation for substantially stable storage of viablethrombocytes, wherein the formulation comprises a pH buffer, ananticoagulant, a non-reducing sugar, a polyol and a functionalizedcarbohydrate. In certain embodiments, the thrombocyte stabilizationformulations comprise a pH buffer, an anticoagulant, a polyol, and afunctionalized carbohydrate. In certain embodiments, the stabilizationformulation comprises a pH buffer, an anticoagulant, a non-reducingsugar, and a functionalized carbohydrate. In still yet anotherembodiment, the stabilization formulation comprises a halogenateddisaccharide derivative and an anticoagulant. In still yet anotherembodiment, the stabilization formulation comprises a halogenateddisaccharide derivative and an anticoagulant furthers comprise a pHbuffer. In certain embodiments, the formulation is one of theformulations set forth in Table 1.

In certain embodiments, the methods comprise admixing a blood samplewith a formulation for substantially stable storage of viable,activatable thrombocytes in a blood sample, wherein the formulationcomprises a pH buffer, an anticoagulant, a non-reducing sugar, a polyol,and a functionalized carbohydrate. In certain embodiments, thethrombocyte stabilization formulation comprises a pH buffer, ananticoagulant, polyol, and a functionalized carbohydrate. In certainother embodiments, the stabilization formulation comprises a pH buffer,an anticoagulant, a non-reducing sugar, and a functionalizedcarbohydrate. In still yet another embodiment, the stabilizationformulation comprises a halogenated disaccharide derivative and ananticoagulant. In still yet another embodiment, the stabilizationformulation further comprises a pH buffer. In certain embodiments, theformulation is one of the formulations set forth in Table 1.

Blood collection tubes, bags, containers and vessels are well-known inthe art and have been employed by medical practitioners for decades.Blood collected for substantially stable storage of one or more bloodcomponent may be obtained from a subject, donor, or patient using anymethod or apparatus commonly employed by those skilled in the art suchas venipuncture or finger prick. In some embodiments, when the blood iscollected by venipuncture, a formulation described herein is locatedinside the blood collection tube, e.g., an evacuated tube (Vacutainer,Becton Dickenson or Vacuette, Greiner) at the time that the blood sampleis obtained from the donor or patient. In some embodiments, thestabilization formulation is added to an already obtained whole bloodsample, preferably immediately or shortly after it is withdrawn.

In some embodiments, the methods described herein use the articles ofmanufacture and kits disclosed.

The following Examples are presented by way of illustration and notlimitation.

Example 1: Stabilization of Inactive Thrombocytes in a Human BloodSample for a Period of at Least 22 Hours at Ambient Temperature

This Example describes formulations of the present invention forstabilizing inactivated thrombocytes that remain capable of beingactivated after being stored for a period of 22 hours at ambienttemperatures.

Whole blood samples were collected from six human donors usingcommercially available hirudin-coated collection tubes (RocheDiagnostics), the blood samples were pooled and within three hours ofcollection, blood samples were processed. A 300 μL aliquot of each wholeblood sample was transferred to an Eppendorf tube at a 1:20 ratio with15 μL of stabilizer formulation A, B, C, or D of Table 1, either priorto or following the addition of the stabilizer formulation, and themixtures were kept at ambient temperatures for predetermined timeperiods before being analyzed. An equal volume of whole blood was addedto each control sample, and each sample was stored at room temperaturein the absence of the stabilizer formulation and processed in parallelwith the test samples.

To 300 μL of each mixture and control, 300 μL of NaCl 0.9% and 20 μL ofthe provided ADP solution was added to promote activation of thethrombocytes and the samples were analyzed using a multiplate analyzer(Roche Diagnostics). Thrombocyte activity in each condition was measuredimmediately after sample set-up (Time 0) using the multiplate analyzerand ADP test according to the manufacturer's instructions. Thrombocyteactivity was also measured at 3 hour, 6 hour, 9 hour and 22 hour timepoints. Thrombocyte activity in each condition was normalized to itsTime 0 measurement. Data from the six donors were then averaged. Thedata are shown in Table 2.

TABLE 2 Stabilization of Viable, Activatable Thromobocytes in a HumanBlood Sample for at Least 22 Hours Time Formulation FormulationFormulation Formulation (hr) Control A B C D 0 100* 100 100 100 100 3 93110 112 96 98 6 82 107 111 101 95 9 77 115 114 101 93 22 59 91 92 74 90*Values are shown as present activity remaining relative to Time 0

As shown in Table 2, after the 9 hour incubation period, the meandecrease in thrombocyte activity in the NF control condition was −23%compared with +15%, +14%, +1% and −7% for Formulations A, B, C, and D ofTable 2, respectively. After a 22 hour incubation period, significantthrombocyte activity was still detected with a mean decrease inthrombocyte activity in the NF control condition of −41%, compared with−9%, −8%, −26% and −10% for Formulations A, B, C, and D of Table 2,respectively.

Formulations of Table 1 comprising a halogenated disaccharidederivative, sucralose, were characterized as described above, and alsowere identified as possessing stabilizing thrombocyte activity in wholeblood for at least 22 hours (Table 3). In this study, theseformulations, as set forth in Table 1, were incorporated into hirudinvacuum blood collection tubes prior to blood draw.

TABLE 3 Stabilization of Viable, Activatable Thromobocytes in a HumanBlood Sample for at Least 22 Hours Formulation Time 0 3 Hours 6 Hours 9Hours 22 Hours Control 100 89 69 58 60 E 100 100 99 98 97 F 100 94 87 8073 *Values are shown as present activity remaining relative to Time 0

During room temperature blood incubation, thrombocyte activity in the NFcontrol condition decreased by −40% by the 22 hour time point, comparedwith −3% for Formula E and −27% for Formula F of Table 1.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising,” which is used interchangeablywith “including,” “containing,” or “characterized by,” is inclusive oropen-ended language and does not exclude additional, unrecited elementsor method steps. The phrase “consisting of” excludes any element, step,or ingredient not specified in the claim. The phrase “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristics of the claimed invention. The present disclosurecontemplates embodiments of the invention compositions and methodscorresponding to the scope of each of these phrases. Thus, a compositionor method comprising recited elements or steps contemplates particularembodiments in which the composition or method consists essentially ofor consists of those elements or steps.

Reference throughout this specification to “one embodiment” or “anembodiment” or “an aspect” means that a particular feature, structure orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present invention. Thus, theappearances of the phrases “in one embodiment” or “in an embodiment” invarious places throughout this specification are not necessarily allreferring to the same embodiment. Furthermore, the particular features,structures, or characteristics may be combined in any suitable manner inone or more embodiments.

The various embodiments described above can be combined to providefurther embodiments. These and other changes can be made to theembodiments in light of the above-detailed description. In general, inthe following claims, the terms used should not be construed to limitthe claims to the specific embodiments disclosed in the specificationand the claims, but should be construed to include all possibleembodiments along with the full scope of equivalents to which suchclaims are entitled. Accordingly, the claims are not limited by thedisclosure.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1-42. (canceled)
 43. A method for stabilizing at least one component ofa blood sample at ambient temperatures, the method comprising: admixinga blood sample from a subject with a composition to form a mixture,wherein the composition comprises a halogenated disaccharide derivativeat about 0.1 μM to about 50 μM and an anticoagulant; and storing themixture at ambient temperatures for at least six hours.
 44. The methodof claim 43, wherein the halogenated disaccharide derivative is a di- ortri-chlorinated disaccharide.
 45. The method of claim 43, wherein thehalogenated disaccharide derivative is sucralose(1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside);trichloronated maltose;1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-6-O-monododecanoate-α-D-galactopyranoside;dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-6-O-monotetradecanoate-α-D-galactopyranoside;or any combinations thereof.
 46. The method of claim 43, wherein theanticoagulant is selected from the group consisting ofethylenediaminetetraacetic acid (EDTA), hirudin, heparin, and sodiumcitrate, or any combination thereof.
 47. The method of claim 43, whereinthe composition further comprises a pH buffer comprising Tris-HC1,citric acid, or 2X phosphate buffered saline.
 48. The method of claim43, wherein the composition further comprises a polyol selected from thegroup consisting of glycol, glycerol, erythritol, threitol, arabitol,xylitol, ribitol, adonitol, mannitol, sorbitol, galactitol, fucitol,iditol, and inositol, or any combinations thereof.
 49. The method ofclaim 48, wherein the polyol is present at a concentration ranging fromabout 90 mM to about 2.5 M.
 50. The method of claim 43, wherein thecomposition further comprises a functionalized carbohydrate selectedfrom the group consisting of sucralfate and sucrose octasulfate, or acombination thereof.
 51. The method of claim 43, wherein the compositionfurther comprises a non-reducing sugar selected from the groupconsisting of sucrose and trehalose, or a combination thereof.
 52. Themethod of claim 43, wherein the pH of the composition is in the range ofabout 4.0 to about 9.0.
 53. The method of claim 43, wherein the at leastone component of a blood sample is one or more of a cell, a nucleic acidmolecule, a peptide or polypeptide, or any combination thereof.
 54. Acomposition for stabilizing at least one component of a blood sample atambient temperatures, the composition comprising: (i) a pH buffer; (ii)an anticoagulant; and (iii) a halogenated disaccharide derivative;wherein the pH of the composition is in the range of about 4.0 to about9.0.
 55. The composition of claim 54, wherein the pH buffer comprisesTris-HCl, citric acid, or 2X phosphate buffered saline.
 56. Thecomposition of claim 54, wherein the anticoagulant is selected from thegroup consisting of ethylenediaminetetraacetic acid (EDTA), hirudin,heparin, and sodium citrate, or any combination thereof.
 57. Thecomposition of claim 54, wherein the halogenated disaccharide derivativeis a di- or tri-chlorinated disaccharide.
 58. The composition of claim54, wherein the halogenated disaccharide derivative is sucralose(1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-α-D-galactopyranoside);trichloronated maltose;1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-6-O-monododecanoate-α-D-galactopyranoside;1,6-dichloro-1,6-dideoxy-β-D-fructofuranosyl-4-chloro-4-deoxy-6-O-monotetradecanoate-α-D-galactopyranoside; or any combinations thereof.
 59. Thecomposition of claim 54, wherein the composition further comprises apolyol selected from the group consisting of glycol, glycerol,erythritol, threitol, arabitol, xylitol, ribitol, adonitol, mannitol,sorbitol, galactitol, fucitol, iditol, and inositol, or any combinationsthereof.
 60. The composition of claim 60, wherein the polyol is presentat a concentration ranging from about 90 mM to about 2.5 M.
 61. Thecomposition of claim 54, wherein the composition further comprises afunctionalized carbohydrate selected from the group consisting ofsucralfate and sucrose octasulfate, or a combination thereof.
 62. Thecomposition of claim 54, wherein the composition further comprises anon-reducing sugar selected from the group consisting of sucrose andtrehalose, or a combination thereof.
 63. A blood collection tubecomprising the composition of claim 54.